Etiological Diagnosis of Atypical Pneumonias

Najčešći uzročnici atipičnih pneumonija su Mycoplasma pneumoniae, Chlamydophila pneumoniae i Legionella pneumophila, rjeđe Chlamydophila psittaci i Coxiella burnetii. Atipične pneumonije mogu biti uzrokovane i različitim virusima. Kliničke i radiološke značajke atipičnih pneumonija nisu specifi čne i defi nitivna dijagnoza ovisi o laboratorijskim testovima. Za postavljanje specifi čne dijagnoze rabe se kultivacija uzročnika, serološke metode, izravno određivanje antigena i molekularne metode. Određivanje etiologije atipičnih pneumonija primarno se temelji na serologiji. Serološkim testiranjem dijagnoza se obično postavlja retrospektivno. Visoki titar protutijela ili četverostruki ili veći porast titra između akutnog i rekonvalescentnog seruma smatra se dijagnostičkim u pacijenata s prisutnim kliničkim simptomima atipične pneumonije. Nijedan dijagnostički test samostalno ne ispunjava sva očekivanja za postavljanje dijagnoze, budući da serološki odgovor može biti različit, kultivacija je zahtjevna i dugotrajna, a molekularne metode nisu standardizirane. Istodobnom uporabom različitih metoda, neodgodivim testiranjem akutnog, a zatim parnog seruma, određivanjem antigena i ako je moguće kultivacijom uzročnika, etiologija atipičnih pneumonija može se defi nirati brže i sigurnije. Komunikacija i suradnja kliničara i mikrobiologa osnovne su za postizanje zadovoljavajućih rezultata. Glavni razlozi za određivanje etiologije atipičnih pneumonija su potvrda adekvatno ordinirane empirijske terapije, rasvjetljavanje epidemiologije i kontrola epidemija.The most common agents associated with atypical pneumonias are Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Chlamydophila psittaci and Coxiella burnetii have been rarely reported. Atypical pneumonia could be also caused by a variety of viral agents. The clinical and radiological features of atypical pneumonia are not specifi c, and diagnosis depends on laboratory tests. Laboratory tests available for specifi c diagnosis include culture, serology, direct antigen detection and molecular methods. The etiology of atypical pneumonia is primarily based on serology. Defi nitive diagnosis through serologic testing is usually retrospective. Either a high initial titer or a fourfold or greater rise between the acute and convalescent serum is considered diagnostic in patients with clinical symptoms of atypical pneumonia. Serologic response can differ, cultures are cumbersome and time-consuming, and molecular methods are not standardized. Therefore, none of the available diagnostic tests fulfi ls all expectations in a diagnosis of atypical pneumonia. Only the simultaneous combination of different methods, immediate testing of acute and later of paired sera, antigen detection and, if possible, cultivation of pathogens, can defi ne the etiology of atypical pneumonia more promptly and accurately. Communication and cooperation among clinicians and microbiologists is essential for successful results. The key reason to make a defi nitive etiological diagnosis of these atypical pathogens is to approve the prescription of empiric therapy, clarify the epidemiology of the disease, and control disease outbreaks

Human granulocytic anaplasmosis in Croatia and new insights about anaplasma and ehrlichia species

Anaplasma phagocytophilum je emergentni patogen kojeg u Europi prenose krpelji Ixodes ricinus koji su vektori i za virus krpeljnog encefalitisa, Borrelia sensu lato, Babesia i neke vrste Rickettsia spp. A. phagocytophilum je obvezno unutarstanična Gram-negativna bakterija koja ima tropizam za granulocite i uzrokuje humanu granulocitnu anaplazmozu (HGA). Dijagnoza HGAtemelji se na kliničkoj procjeni i mora se potvrditi serološki dokazom serokonverzije ili četverostrukog porasta titra protutijela IgG ili određivanjem DNK. Protutijela su često negativna u početnoj fazi akutne bolesti i obavezno treba testirati parne serume u razmaku od 2–4 tjedna. U ranoj fazi bolesti kada je serologija još negativna značajniji je nalaz DNK iz uzorka krvi s antikoagulansom, ali dostupnost PCR je ograničena. Morule u razmazu periferne krvi obojene po Giemsi značajne su za rano postavljanje dijagnoze, ali se u HGA pronalaze jako rijetko. Od 2009. do 2012.g. u Klinici za infektivne bolesti u Zagreb testirali smo protutijela IgM i IgG protiv A. phagocytophilum u 496 seruma od 425 bolesnika. Parne serume imalo je samo 68 bolesnika. Pozitivna protutijela na A. phagocytophilum nađena su u 160 (37,6%) bolesnika. Tri bolesnika su zadovoljila kriterije za akutnu HGA. Prisutnost IgG u titru 256 ili većem, što se definira kao moguća HGA, nađena je u 40 bolesnika. Samo IgM imalo je 17, a IgM i IgG anti-A. phagocytophilum 16 bolesnika. Anti-A. phagocytophilum IgG u titru 64 ili 128 imalo je u 84 bolesnika. Podaci potvrđuju prisutnost infekcije A. phagocytophilum u Hrvatskoj premda većina akutnih infekcija ostaje nedokazana, većinom kao samoizlječive bolesti. HGA se treba uključiti u diferencijalnu dijagnozu bolesnika sa simptomima sličnima gripi u područjima gdje se nalazi Ixodes ricinus, posebno u vrijeme aktivnosti krpelja. U životinja i krpelja u Hrvatskoj utvrđena je prisutnost i drugih patogenih članova porodice Anaplasmataceae zbog čega dodatno treba misliti o HGA uz veća dijagnostička nastojanja za postavljanje dijagnoze.Anaplasma phagocytophilum is an emergent tick-born pathogen in Europe transmitted by Ixodes ricinus ticks which may also transmit tick-borne encephalitis virus, Borrelia sensu lato, Babesia and some Rickettsia spp. A. phagocytophilum is an obligate intracellular Gram- negative bacteria that has tropism for granulocytes and causes human granulocytic anaplasmosis (HGA). The diagnosis of HGA relies on clinical suspicion and must be confirmed with seroconversion or 4-fold increase in antibody titre or by DNA determination. Antibodies are often negative in the initial phase of acute illness and paired sera taken 2–4 weeks later are obligatory. PCR on anticoagulated blood could be more efficient tool in this phase but it is of limited availability. Morulae in Giemsa-stained peripheral blood smears may provide early diagnosis but could be observed very rarely in HGA. From 2009 till 2012, we tested 496 sera from 425 patients for IgM and IgG anti-A. phagocytophilum antibodies at the University Hospital for Infectious Diseases in Zagreb. Paired sera were sent for only 68 patients. Positive antibodies against A. phagocytophilum were found in 160 (37.6%) patients. Three patients fulfilled the criteria for acute HGA. The presence of IgG in titres 256 or higher defined as probable HGA was found in 40 patients. Only IgM, and IgM and IgG anti-A. phagocytophilum antibodies were nadetected in 17 and 16 patients, respectively. Anti-A. phagocytophilum IgG in titres 64 or 128 was found in 84 patients. The data show that A. phagocytophilum infections are present in Croatia, although most of the acute infections remain unconfirmed as self-resolved diseases. HGA should be included in the differential diagnosis of patients with flu-like illness in regions with Ixodes ricinus, especially during tick-activity season. Different members of Anaplasmataceae family were found in animals and ticks in Croatia, which is why better awareness of HGAand diagnostics effort are needed

Point-of-care (POC) testing in diagnostics of infectious diseases

Razvojem tehnologija laboratorijskih testova zadnjih se godina značajno povećao broj dostupnih testova za dijagnostiku infektivnih bolesti koji se mogu izvoditi izvan laboratorija: uz bolesničku postelju, u ambulanti, savjetovalištu i drugim nelaboratorijskim uvjetima. Takav oblik testiranja naziva se point-of-care (POC) testiranje – testiranje na mjestu gdje se bolesniku pruža skrb. POC testiranje je laboratorijsko ispitivanje koje se provodi jednostavnim sustavom mjerenja načelno izvan laboratorija u zdravstvenim ustanovama za bolničke i izvanbolničke bolesnike. POC testiranje provodi osoblje koje nema primarnu laboratorijsku osposobljenost i nema iskustvo u laboratorijskoj medicini (medicinske sestre i tehničari, liječnici). Prije uvođenja POC testiranja neophodno je evaluirati potrebu i svrhu uvođenja ovih testova kao zamjene za rutinski laboratorijski rad. Potrebno je definirati broj testiranja, metodu i cijenu kao i tko će i za koju namjenu koristiti POC testove te kada i kako će se testiranje izvoditi. Uvođenje POC testiranja ne smije biti stihijsko. POC testiranje treba planirati u dogovoru s nadležnim laboratorijem koji će provoditi kontrolu kvalitete rada. Osoblje koje će raditi testiranje treba prethodno dobro educirati. Iako se radi o jednostavnim testovima, svako testiranje mora zadovoljavati pravila dobre laboratorijske prakse budući da se ne radi o tzv. kućnim testovima, već o testovima rezultate kojih treba priopćiti bolesniku i adekvatno sačuvati. POC testove ima smisla raditi samo ako rezultat izravno utječe na odluku o daljnjem postupku s bolesnikom. Neispravno korištenje POC testova predstavlja rizik za bolesnika, a istovremeno predstavlja dodatni povećani trošak zdravstvene skrbi. Jedino primjena klinički evaluiranih testova odobrenih za korištenje može značajno unaprijediti kvalitetu obrade bolesnika u cilju dobivanja što bržeg i točnijeg nalaza koji će usmjeriti daljnji postupak, a ujedno smanjiti troškove dijagnostičke obrade.Technological development of laboratory tests in recent years significantly increased the number of tests available for the diagnosis of infectious diseases that can be conducted outside the laboratory: near the bedside, in the clinics, counseling centers and other non-laboratory conditions. Such a form of testing is called point-of-care (POC), testing where the patient care is provided. POC testing is a laboratory testing carried out using a simple measuring principle outside the laboratory in medical institutions for hospital and ambulatory patients. POC testing is performed by personnel with no primary laboratory qualifications and no experience in laboratory medicine (nurses, technicians, doctors). Before the introduction of POC testing it is necessary to evaluate the need for and purpose of the introduction of these tests as a substitute for routine laboratory work. It is necessary to define the number of testing, method and cost as well as who and for what purpose to use POC test, and when and how it will be run. Introducing POC testing should not be haphazard. POC testing should be planned in agreement with a relevant laboratory that will conduct a quality control. Personnel who will do the testing should be thoroughly trained. Although these are simple tests, all testing must comply with the rules of good laboratory practice because these are not "home tests" but the tests the results of which should be communicated to the patient and adequately saved. POC tests are worth doing only if the result will directly affect the decision on how to proceed with the patient. Incorrect use of POC tests presents a risk to patient and an additional increased cost of health care. Only the use of clinically evaluated test approved for use can significantly improve the quality workup of the patient obtaining the rapid and accurate findings which will direct further treatment, and also reduce the additional costs of diagnostic examination

Pitfalls and Benefits of Serological Diagnosis of Lyme Borreliosis From a Laboratory Perspective

Lajmska borelioza (LB) je bolest koju u Europi najčešće uzrokuju borelije kompleksa Borrelia burgdorferi sensu lato, dok je u Sjevernoj Americi jedino patogena B.burgdorferi sensu stricto. Kliničke manifestacije LB su polimorfne. Postavljanje dijagnoze temelji se na kliničkoj slici i epidemiološkim podacima o vjerojatnosti kontakta s krpeljima uz primjenu mikrobiološke dijagnostike. Najčešća je rutinska dijagnostika serologija za određivanje i praćenje dinamike specifičnih protutijela IgM i IgG. Nakon primarnog testiranja, rezultate je potrebno potvrditi dodatnim testom visoke specifičnosti. U područjima visoke prevalencije, specifičnost rezultata visoko osjetljivog i specifičnog testa nije obvezno dodatno potvrđivati. I pozitivni i negativni nalazi moraju se interpretirati klinički. Serološka dijagnostika predstavlja dobrobit za prepoznavanje i liječenje bolesnika samo ako se rezultati interpretiraju temeljem poznavanja patogeneze, kliničke slike i imunosnog odgovora kao i karakteristika korištenog testa. Karakteristike samih borelija i prezentacije antigena, izbjegavanje imunosnog odgovora, dostupnost različitih testova kao i Interneta predstavljaju zamke, posebno ako se slijede neprovjerene informacije.Lyme borreliosis (LB) in Europe is most commonly caused by different borrelia of Borrelia burgdorferi sensu lato complex , while in North America the only pathogen is B.burgdorferi sensu stricto. Clinical manifestations of LB are polymorphic. Diagnosis is based on the clinical presentation and epidemiological data on the likelihood of contact with ticks and with the application of microbiological diagnostics. The most common routine diagnosis is serology to determine and monitor the dynamics of specific IgM and IgG antibodies. After primary testing, the results need to be confirmed by an additional high-specificity test. In regions with a high prevalence, the specificity of the results of a highly sensitive and specific test does not need to be further confirmed. Both positive and negative findings must be interpreted clinically. Serological diagnosis is beneficial for the recognition and treatment of patients only if the results are interpreted on the basis of knowledge of the pathogenesis, clinical presentation and immune response as well as the characteristics of the test used. The characteristics of borrelia itself and the presentation of the antigen, the avoidance of the immune response, the availability of various tests as well as the Internet are pitfalls, especially if unverified information is followed

SPECIFICITY OF LYME NEUROBORRELIOSIS DIAGNOSTICS

Lajmska neuroborelioza (LNB) nastaje hematogenim rasapom borelija u središnji živčani sustav (SŽS), a opisan je i prodor putem perifernog živca. Razvija se serozni meningitis sa ili bez pareze kranijalnog živca što je prevladavajuća klinička slika. Najčešće je zahvaćen n. facialis. Za razliku od Sjeverne Amerike, u Europi mogu biti zahvaćeni i drugi kranijalni živci, što se povezuje s prevaliranjem različitih vrsta borelija - u Europi najčešće B. garinii, B. bavariensis i B. afzelii, a u Sjevernoj Americi samo B. burgdorferi sensu stricto. Meningoradikulitis ili Bannwarthov sindrom tipična je slika LNB samo u Europi. Simptomi LNB većinom nisu tipični i mogu sličiti različitim neurološkim bolestima, pa dijagnozu često nije jednostavno definirati. Dijagnostika LNB mora uključivati analizu likvora u kojem je značajan nalaz pleocitoze, što upućuje na serozni meningitis kojem je potrebno dokazati povezanost s borelijama. Mikrobiološka dijagnostika LNB obuhvaća kultivaciju, zahtjevnu i dugotrajnu metodu (9-12 tjedana) koja se radi isključivo u referentnim centrima, te molekularnu (PCR) i serološku dijagnostiku. Zbog malog broja borelija u likvoru kao i relativno male količine likvora koja se šalje za analizu, molekularna je dijagnostika često lažno negativna. Stoga je serološka dijagnostika ključna za dokazivanje LNB. Serologija se radi iz istovremeno uzetih uzoraka seruma i likvora u kojima se određuju specifična protutijela IgM i IgG te ukupni imunoglobulini i/ili albumini. Serum i likvor moraju se analizirati istom metodom u istim uvjetima kako bi se mogla odrediti intratekalna sinteza specifičnih protutijela, tj. izračunati indeks protutijela (antibody index, AI). Specifična protutijela u lajmskoj boreliozi nastaju relativno sporo, a s duljinom trajanja infekcije njihova količina se povećava. U ranoj LNB protutijela u likvoru ne može se uvijek otkriti, iako je prisutna pleocitoza. Kasnu LNB u pravilu prati jaki imunosni odgovor i uz pleocitozu često se nalazi pozitivan AI. Nakon infekcije, vremenom se likvor normalizira i pleocitoza se više ne nalazi, iako specifična protutijela u likvoru mogu ostati dugo prisutna, čak i uz pozitivan AI. Stoga je potrebno pratiti imunosni odgovor u krvi i likvoru od dana kada se bolesnik javi zbog simptoma i zatim nakon npr. jednog, tri, šest i dvanaest mjeseci, radi procjene korelacije nalaza i bolesti. Dijagnoza LNB mora biti u skladu s kliničkim, epidemiološkim i anamnestičkim podatcima te laboratorijskim nalazima, posebno u likvoru. LNB je 1) potvrđena ako uz kliničku sliku postoji pleocitoza i intratekalna sinteza specifičnih protutijela; 2) vjerojatna ako intratekalna sinteza nije potvrđena, a specifična protutijela su prisutna u krvi bolesnika; 3) LNB nije vjerojatna ako nema pleocitoze ili nema analize likvora, iako su prisutna specifična protutijela u krvi bolesnika, ali klinička slika i epidemiološka anamneza nisu karakteristični. CXCL13 je biljeg koji može biti koristan kao dodatni test premda nije specifičan za LNB - u likvoru je povišen i prati akutnu upalu. Ako postoji mogućnost, LNB bi trebalo potvrditi kultivacijom i molekularnom dijagnostikom. Interpretacija laboratorijskih i kliničkih nalaza zahtijeva znanje i iskustvo. Informacije se trebaju sagledati u skladu s okolnostima i specifičnosti bolesnika, tako da je svaki bolesnik poseban dijagnostički izazov.Lyme neuroborreliosis (LNB) is caused by hematogenous spread of Borrelia into the central nervous system (CNS), but entry through a peripheral nerve has also been described. Aseptic meningitis develops with or without cranial nerve palsy, which is the predominant clinical presentation. Facial nerve is most frequently affected. Unlike North America, other cranial nerves can be affected in Europe, which is related to the prevalence of different types of Borrelia. Borrelia (B.) garinii, B. bavariensis, and less frequently B. afzelii are most common in Europe, while B. burgdorferi sensu stricto is the only North American strain. Meningoradiculitis or Bannwarth syndrome is a typical LNB presentation described only in Europe. The symptoms of LNB can resemble various neurological diseases, which makes the diagnosis of LNB difficult. The diagnosis of LNB must include analysis of cerebrospinal fluid (CSF) in which pleocytosis is significant to support aseptic meningitis, and the association with Borrelia must be proven. Microbiological diagnosis of LNB includes cultivation, a demanding and long-term method (9-12 weeks), which is performed exclusively in reference centers, and molecular (polymerase chain reaction, PCR) and serological diagnostics. PCR is often false-negative due to the low number of strands in the CSF. Thus, serological diagnosis remains crucial to confirm LNB. Serology is performed on simultaneously collected serum and CSF samples, from which specific IgM and IgG antibodies, total immunoglobulins and/or albumins need to be determined. Serum and CSF samples must be analyzed by the same method under the same conditions in order to assess the intrathecal synthesis of specific antibodies, i.e., to calculate the antibody index in CSF (antibody index, AI). In patients with Lyme borreliosis, specific antibodies are produced relatively slowly, and their quantity increases with the duration of the infection. In early LNB, antibodies in the CSF are not always detectable while pleocytosis is present. In late LNB, a strong immune response is present in the CSF, as well as pleocytosis, and a positive AI can be determined. Over time, CSF normalizes and pleocytosis is no longer detected, but CSF antibodies can remain present for a long period of time. Therefore, the immune response in the blood and CSF has to be monitored from the day the patient presented with symptoms, and then, for example, at one, three, six and twelve months to assess the correlation of laboratory findings with the disease. The diagnosis of LNB must be in accordance with clinical, epidemiological and history data and laboratory findings, especially in CSF. LNB is confirmed if the clinical picture is accompanied by pleocytosis and intrathecal synthesis of specific antibodies; LNB is probable if intrathecal synthesis is not confirmed while specific antibodies are present in the patient’s blood; and LNB is unlikely if there is no pleocytosis or no CSF analysis, although specific antibodies are present in the patient’s blood but the clinical picture and epidemiological history are not characteristic. If there is a possibility, LNB should be confirmed by culture and molecular diagnostics. CXCL13 is a marker that can be useful as an additional test, even though it is not specific for LNB as it is elevated in CSF and observed during acute inflammation. Interpretation of laboratory and clinical findings in LNB requires knowledge and experience. The findings should be interpreted in accordance with the circumstances and condition of the patient, and therefore each patient represents a special diagnostic challenge

HEPATITIS E IN CROATIA – GUIDELINES FOR DIAGNOSIS AND TREATMENT

Hepatitisu E pridaje se velika pozornost kao emergentnoj zoonozi koja se pojavljuje diljem svijeta. Kliničke slike variraju od teških fulminantnih oblika u nerazvijenim zemljama do blažih, dijagnostički neprepoznatih hepatitisa u razvijenima. Sve se češće opisuje kronični hepatitis E u osoba s transplantiranim solidnim organima i HIV-bolesti. Dijagnoza hepatitisa E postavlja se određivanjem protutijela anti-HEV-IgM i IgG uz potvrdni Western blot te dokazivanjem HEV RNK. U Hrvatskoj je prvi slučaj autohtone bolesti dokazan 2012. godine uzrokovan HEV-om genotipa 3 (HEV-3). Prikazujemo suvremene spoznaje o hepatitisu E, nadopunjene vlastitim rezultatima testiranja u Klinici za infektivne bolesti u Zagrebu te smjernice za dijagnostiku i liječenje imunokompetentnih i imunokompromitiranih osoba. Od 2011. do 2014. godine HEV je testiran u 1107 bolesnika, a anti-HEV-protutijela imalo je njih 117 (10,6%). Akutna HEV-infekcija dijagnosticirana je u 25 (2,3%) bolesnika. S obzirom na utvrđenu prevalenciju protutijela, zaključujemo da HEV-dijagnostika treba biti dio dijagnostičkog panela za bolesnike s povišenim aminotransferazama.Hepatitis E is attributed great attention as an emerging worldwide-distributed zoonosis. The clinical presentation varies from severe fulminant in underdeveloped to milder forms of diagnostically unrecognized hepatitis cases in developed countries. Chronic hepatitis E is more often described in subjects with transplanted solid organs and HIV disease. The diagnosis of hepatitis E is established by determination of anti-HEV IgM and IgG with Western blot confirmation and detection of HEV RNA. In Croatia, the first case of indigenous disease caused by HEV genotype 3 (HEV-3) was detected in 2012. In this paper we present current knowledge on hepatitis E supplemented by own results obtained at the University Hospital for Infectious Diseases in Zagreb and guidelines for diagnosis and treatment of immunocompetent and immunocompromised individuals. In the period from 2011-2014, HEV was tested in 1107 patients, of whom 117 (10.6%) had anti-HEV antibodies. Acute HEV infection was diagnosed in 25 (2.3%) patients. Considering the prevalence of antibodies we can conclude that HEV diagnostics should be included in the diagnostic panel for patients with elevated aminotransferases

Enzyme immunoassay for separate detection of anti-HCV antibodies to individual HCV antigens as a confirmatory assay in diagnostics of HCV infection

Dijagnostika hepatitisa C temelji se na određivanju protutijela anti-HCV probirnim imunoenzimskim testovima (EIA). Svaki reaktivni nalaz anti-HCV probirnog testa prema recentnim smjernicama zahtijeva daljnje određivanje HCV RNK. U slučajevima negativnog nalaza HCV RNK, reaktivni nalaz EIA anti-HCV treba potvrditi imunoblot testom (IB). Komercijalno su dostupni i EIA testovi koji imaju mogućnost određivanja specifičnih protutijela na pojedinačne antigene HCV koji daju rezultate usporedive sa standardnim potvrdnim IB testovima. Cilj ovog rada bio je prikazati EIA potvrdni test, EIA-Anti-HCV-Spectrum (DSI, Italija) i usporediti rezultate testiranja sa standardnim IB testom. Analizirali smo 50 nasumično odabranih seruma koji su imali reaktivni odgovor protutijela na HCV u probirnom EIA testu (Monolisa HCV Ag-Ab ULTRA, Bio-Rad, Francuska). Za isključenje lažno pozitivnih rezultata serumi su dalje testirani potvrdnim IB testom (Deciscan HCV PLUS, Bio-Rad, Francuska). Odabranih 50 uzoraka testirano je testom EIA-Anti-HCV-Spectrum za određivanje pojedinačnih protutijela anti-HCV na antigene jezgre te antigene NS3, NS4 i NS5. Od 50 seruma s reaktivnim odgovorom protutijela na HCV u probirnom testu u oba potvrdna testa pozitivno je bilo 40, negativno 1 i granično 3 uzorka. Nepodudarnih rezultata potvrdnih testova bilo je 6: svi su bili granično pozitivni u IB testu, a negativni u alternativnom potvrdnom testu EIA-Anti-HCV-Spectrum. Predstavili smo imunoenzimski potvrdni test za protutijela anti-HCV koji je pokazao dobru korelaciju sa standardnim imunoblot testom premda je potrebno napraviti evaluaciju na ve}em broju uzoraka za sigurnije zaključke.Diagnostics of hepatitis C is based on the determination of anti-HCV antibodies by screening enzyme immunoassays (EIA). Each reactive anti-HCV screening result, according to recent guidelines, requires further determination of HCV RNA. In cases of negative HCV RNA findings, reactive anti-HCV results should be confirmed by immunoblotting (IB). Alternative confirmatory EIA with the ability to determine the antibodies against individual HCV antigens, which are comparable with the results of immunoblot tests, are commercially available. The aim of this study was to present an EIA test, the EIA-Anti-HCV-Spectrum (DSI, Italy) and compare the test results with standard IB test. We analyzed 50 randomly selected serum samples with reactive screening EIA (Monolisa HCV Ag-Ab ULTRA, Bio-Rad, France) results. In order to exclude false positives, serum samples were further tested with confirmatory IB test (Deciscan HCV PLUS, Bio-Rad, France). A total of 50 selected samples were tested with EIA-Anti-HCV-Spectrum for separate detection of anti-HCV antibodies against core, NS3, NS4 and NS5 antigens. Out of the 50 primary reactive samples, anti-HCV antibodies were positive in both confirmatory tests in 40, negative in 1 and borderline in 3 samples. Discordant results of confirmatory tests were recorded in 6 samples: all borderline in IB test and negative in the alternative EIA-Anti-HCV-Spectrum test. We present an EIA confirmatory test for anti-HCV antibodies which showed a good correlation with the standard immunoblot test, although it is ecessary to perform the evaluation on a large number of samples for safer conclusions

RECENT DEVELOPMENTS IN SEROLOGIC AND MOLECULAR DIAGNOSIS OF HEPATITIS B AND C

Opisali smo glavne novosti u dijagnostici hepatitisa B i C kao dio hrvatskih smjernica za dijagnostiku i liječenje virusnih hepatitisa 2013. Dijagnostika akutnog i kroničnog hepatitisa B započinje određivanjem HBsAg, anti-HBc i anti-HBs. Ostale serološke biljege hepatitisa B treba određivati tek u drugom koraku ako su nalazi HBsAg i/ili anti-HBc pozitivni. Pozitivan nalaz samo na anti-HBc potrebno je obvezno nadopuniti određivanjem HBV DNK. Kvantitativno određivanje HBsAg treba se koristiti komplementarno s određivanjem HBV DNK za: (i) razlikovanje inaktivnog nositelja HBsAg od aktivnog kroničnog HBeAg-negativnog hepatitisa B u bolesnika sa HBV DNK nižom od 2.000 IU/mL te za (ii) praćenje tijeka liječenja kroničnog hepatitisa B s pegiliranim interferonom-alfa. Metoda PCR-a u stvarnom vremenu ostaje i dalje metoda izbora za detekciju i kvantifikaciju HBV DNK. Testiranje za HCV započinje određivanjem specifičnih protutijela probirnim enzimskim imunotestovima ili brzim testovima. Sve osobe s pozitivnim probirnim anti-HCV testom treba testirati na prisutnost HCV RNK ili antigena virusne kapside. Potvrdne anti-HCV testove treba koristiti samo kao dodatne testove koji će potvrditi ili isključiti značenje reaktivnih rezultata probirnih enzimskih imunotestova u osoba koje su HCV RNK negativne. U praćenju virusne kinetike tijekom trojne terapije hepatitisa C preporučuje se korištenje molekularnih testova s identičnom donjom granicom detekcije (LLOD) te donjom granicom kvantifikacije. Određivanje rezistencije HCV-a na inhibitore proteaze nije dio obveznog dijagnostičkog praćenja bolesnika liječenih s trojnom terapijom. Ne postoje dostatni dokazi o potrebi uvođenja subtipizacije HCV-a u obveznu predterapijsku obradu bolesnika s kroničnim hepatitisom C. Genotip IL-28 je važan prediktor SVR-a u bolesnika liječenih kombinacijom IFN i ribavirina kao i u bolesnika zaraženih genotipom 1 liječenih trojnom terapijom. Genotipizacija IL-28B preporučuje se u predterapijskoj obradi bolesnika s kroničnim hepatitisom C. Posebno je značajan dijagnostički parametar u terapijski-naivnih bolesnika s kroničnim hepatitisom C prilikom dvojbe dvojna vs. trojna terapija.The 2013 Update of the Croatian Guidelines for the Diagnosis and Treatment of Viral Hepatitis summarizes recent developments in the diagnosis of hepatitis B and C. Determination of HBsAg, anti-HBc and anti-HBs is the initial step in the diagnostic workup of acute and chronic hepatitis B. Other hepatitis B serologic markers should be analyzed in the second stage of the diagnostic workup in HBsAg and/or anti-HBc positive patients. A positive anti-HBc finding should be followed by HBV DNA quantification. HBsAg quantification is complimentary to the HBV DNA quantification and is used: (i) to differentiate between inactive HBsAg carriers and active chronic HBeAg-negative hepatitis B in patients with HBV DNA <2000 IU/mL; and (ii) for treatment monitoring in patients with chronic hepatitis B receiving pegylated interferon-alpha. Real-time PCR remains the method of choice for detection and quantification of HBV DNA. The first step in HCV testing is determination of specific antibodies via screening assays, enzyme immunoassays or point-of-care assays. All persons with positive results of anti-HCV screening assays should be additionally tested for HCV RNA or presence of HCV viral capsid antigen. Confirmatory anti-HCV assays should be used as additional assays for confirmation of reactive results obtained by screening enzyme immunoassays in HCV RNA-negative persons only. Molecular assays with identical lower limit of detection (LLOD) and lower limit of quantification are recommended for monitoring of viral kinetics during chronic hepatitis C triple therapy. HCV resistance testing to protease inhibitors is not part of the recommended diagnostic monitoring of patients receiving triple therapy. HCV subtyping is currently not recommended as part of pretreatment diagnostic algorithm due to currently insufficient evidence on its clinical usefulness. IL-28 genotype is an important predictor of SVR in patients treated with a combination of interferon-alpha and ribavirin as well as in patients with HCV genotype 1 receiving triple therapy. IL-28B genotyping is recommended as part of pretreatment diagnostic workup in patients with chronic hepatitis C and is a particularly important parameter for recommending double versus triple therapy in treatment-naïve patients with chronic hepatitis C

First Case of Q Fever Endocarditis in Croatia and a Short Review

We present a 70-year-old man from Dalmatia, Croatia, with a history of prolonged high fever diagnosed as Q fever endocarditis. As far as we know, this is the first case of chronic Q fever in Croatia. The treatment was started as for culture-negative endocarditis, but was without clinical response. After significantly high anti-phase I IgG plus IgA antibodies titers to Coxiella burnetii were shown, the initial treatment with doxycycline was changed and ciprofloxacin was started with good clinical response

ARE BORRELIA MIYAMOTOI AND BORRELIA MAYONII POSSIBLE PATHOGENS IN CROATIA?

Borrelia miyamotoi pripada grupi borelija koje uzrokuju povratnu vrućicu, a prenosi se istim krpeljima (Ixodidae) kao i Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia spp. te virus krpeljnog meningoencefalitisa. B. miyamotoi dokazana je u malih glodavaca u Hrvatskoj. U Europi bolest nije česta, a prezentira se vrućicom praćenom zimicom, znojenjem, glavoboljom, umorom, mialgijama i artralgijama. U imunokompromitiranih bolesnika bolest može biti teška s meningoencefalitisom. Infekcija B. miyamotoi dijagnosticira se molekularnim testovima nakon isključivanja drugih krpeljima prenosivih bolesti. Serološke studije rađene su testovima temeljenima na rekombinantnom GlpQ proteinu koji nisu komercijalno dostupni. Nakon detaljne kliničke i mikrobiološke evaluacije moguća infekcija B miyamotoi u Hrvatskoj može se dijagnosticirati molekularnim metodama in house. Borrelia mayonii izolirana je samo u SAD-u iz krpelja Ixodes scapularis i u malog broja bolesnika. Nema dokaza za postojanje B. mayonii u Europi. Bolesnici s B. mayonii imali su akutnu febrilnu bolest s osipom i mogućim neurološkim simptomima. Dijagnoza infekcije B. mayonii postavljena je nalazom spirohetemije koja je dokazana kultivacijom i mikroskopiranjem preparata krvnog razmata te metodom PCR. Serološki testovi za detekciju B. mayonii nisu komercijalno dostupni. Preporuke za liječenje infekcije B. miyamotoi i B. mayonii temelje se na objavljenim kliničkim prikazima bolesnika te preporukama za liječenje lajmske borelioze. Nakon provedene terapije bolesnici su se oporavili bez komplikacija.Borrelia (B.) miyamotoi belongs to the group of relapsing fever borrelia and is transmitted by the same ticks (Ixodidae) as Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia species and tick-borne fl aviviruses. B. miyamotoi was detected in small rodents in Croatia. In Europe, B. miyamotoi infections are not common, and mainly present as febrile illness with chills, sweats, headache, fatigue, myalgias and arthralgias. In immunocompromised patients, the disease may be severe with meningoencephalitis. B. miyamotoi diseases have been diagnosed with molecular methods after excluding other tick-borne diseases. Serological assays based on recombinant B. miyamotoi GlpQ protein used in published serological studies are not commercially available. After detailed clinical and microbiological evaluation, the possible B. miyamotoi infection in Croatia can be diagnosed with in-house methods. Borrelia (B.) mayonii has only been detected among few patients and Ixodes scapularis ticks in the United States so far. There is no evidence for B. mayonii in Europe. Patients with B. mayonii infection had febrile illness with rash and probable neurological symptoms. The diagnosis of B. mayonii infection was defi ned by detection of spirochetes in peripheral blood with culture isolation, microscopic smears, and polymerase chain reaction method. Serological tests for B. mayonii are not commercially available. Treatment recommendations for B. miyamotoi and B. mayonii infections are based on the published case reports and recommendations for treatment of Lyme disease. After antimicrobial treatment, all patients recovered without complications